New reference values for maximum respiratory pressures in healthy Brazilian children following guidelines recommendations: A regional study

Objective To determine reference values for maximum static respiratory pressures in healthy children from a Brazilian region, following recommendations of the European Respiratory Society (ERS) and the Brazilian Society of Pneumology and Tisiology (SBPT). Methods A cross-sectional observational study was conducted with healthy children (6 to 11 years) of both sexes. The maximum inspiratory and expiratory pressures (PImax and PEmax, respectively) were measured using a digital manometer. Each child performed a minimum of three and a maximum of five maneuvers; three acceptable and reproducible maneuvers were considered for analysis. Minimum time for each maneuver was 1.5 seconds, with a one-second plateau, and one minute of rest between them. A stepwise multiple linear regression analysis was conducted for PImax and PEmax, considering correlations between independent variables: age, weight, and sex. Results We included 121 children (62 girls [51%]). Boys reached higher values for maximum respiratory pressures than girls. Respiratory pressures increased with age showing moderate effect sizes (PImax: f = 0.36; PEmax: f = 0.30) between the stratified age groups (6–7, 8–9, and 10–11 years). Age and sex were included in the PImax equation (PImax = 24.630 + 7.044 x age (years) + 13.161 x sex; R2 = 0.189). PEmax equations were built considering age for girls and weight for boys [PEmax (girls) = 55.623 + 4.698 x age (years) and PEmax (boys) = 82.617 + 0.612 x weight (kg); R2 = 0.068]. Conclusions This study determined new reference equations for maximal respiratory pressures in healthy Brazilian children, following ERS and SBPT recommendations.


Introduction
Respiratory muscle strength is generally estimated using respiratory pressure measurements, muscle contraction, and changes in lung volumes and chest wall structures [1]. Respiratory pressures can be measured using voluntary maneuvers (e.g., maximum respiratory pressures, sniff nasal inspiratory pressure), or involuntary contractions in response to phrenic nerve stimulation [1,2].
Maximum inspiratory (PImax) and expiratory pressures (PEmax) are non-invasive and straightforward tests commonly used in clinical practice. These measurements are essential in diagnosing respiratory muscles weakness [3]. In neuromuscular diseases (e.g., amyotrophic lateral sclerosis, myotonic dystrophy, and myasthenia gravis), PImax, PEmax, sniff nasal inspiratory pressure, and spirometry values are sensitive indicators of disease severity in the earliest stages [4,5].
Reference values for respiratory pressures vary among studies and may be related to biological characteristics of the participants (i.e., age, sex, weight, height, cultural differences), technique and equipment used, evaluator expertise, participants motivation during the test, methodological procedures, and data analysis [2,6,7].
Studies investigated reference values for PImax and PEmax for healthy Brazilian children [8,9]. However, there is no consensus on the reference values reported by the studies. This may be because each study used a different protocol, equipment (e.g., aneroid manometer) [10], age groups [9,11], and was developed in different country regions.
A lack of assessment standardization led to variations in reference values reported by each study. This variation can lead to the risk of underestimating or overestimating pressure values, with a bias to the correct diagnosis of respiratory disorders and, consequently, delay in starting the required therapy. With new studies that assess reference values, using recommended methodologies, and following methodological rigor, we can support predict the severity of dysfunctions and begin treatment within a suitable therapeutic time frame.
Therefore, this study aimed to establish new reference values for maximum static respiratory pressures in healthy children from a Brazilian region, considering European Respiratory Society (ERS), and Brazilian Society of Pneumology and Tisiology (SBPT) [1,7,12] guidelines. This study also aimed to compare the results, obtained from a digital manometer, with predicted values and reference equations for respiratory pressures in children reported by previous studies. parents received information about the study and signed informed assent and consent forms, respectively.

Study design
A cross-sectional observational study was conducted in Natal/Rio Grande do Norte, Brazil. Healthy children ranging from ages 6 to 11 years old were recruited in private and public schools. Sample size was previously calculated in the study that determined reference values of sniff nasal inspiratory pressure (SNIP) [13]. Spirometry was previously developed to certify eligibility for lung function (FVC and FEV 1 > 80% of predicted and FEV 1 /FVC > 70%) [14]. This group was stratified by sex and age (6-7, 8-9, and 10-11 years old). We included children: (1) with no previous respiratory, cardiac, neurovascular, and neuromuscular diseases; (2) who no had influenza during or one week before the evaluation; (3) with no regular use of allergy medications, corticosteroids, or central nervous system depressants; (4) with no previous surgeries requiring incision in the thoracic or abdominal cavities; and (5) who had the ability to follow instructions or verbal command [12,14,15]. The lower age limit was established due to the risk of error in the reproducibility of the maneuvers, compromising data quality.
Initially, weight and height was assessed using a mechanical scale coupled to a stadiometer (Model 110-CH, Welmy, Brazil). The percentile for body mass index was calculated using WHO Anthro Plus 1 v3.2.2 software (World Health Organization, Switzerland). Spirometry was performed using a calibrated Koko spirometer (nSpire Health Inc, Longmont, CO, USA), with children in sitting position, as recommended by ERS [14]. Spirometry data were interpreted according to the values predicted by Mallozi published by Pereira [16]. PImax and PEmax were obtained using a digital manometer (NEPEB-LabCare/UFMG, Brazil). Data were processed by Manovac software (version 4.1), which provides mean, peak, and plateau pressure values. Procedures followed recommendations of the ERS [1] and SBPT [12], attempting to minimize errors and differences due to methodological variations between previous studies. Tests were explained, demonstrated, and conducted by the same evaluator. Children were in sitting position, with feet and trunk supported, and wearing nose clips. A disposable cylindrical mouthpiece with a leakage escape of approximately 2 mm was used to prevent glottic closure at PImax, and to minimize pressures generated by orofacial muscles and air leak during PEmax. The evaluator held hands to children's cheeks during maneuvers to prevent air leakage. PImax was performed after a maximum expiration (i.e., close to residual volume). PEmax was performed after a maximum inspiration (i.e., close to total lung capacity). Minimum time for each maneuver was 1.5 seconds with one second plateau, and one minute of rest between each maneuver. Each child performed a minimum of three and a maximum of five maneuvers to achieve three acceptable (without air leak and adequate duration) and reproducible maneuvers (values of two largest maneuvers could not differ more than 10%, and value of the third largest maneuver could not differ more than 20% from the highest value) [7]. Last maneuver could not be the greatest since it would indicate learning effect. When this occurred, evaluation should continue until a lower value was reached [7]. Fig 1 summarizes the procedures performed. The highest value of the maximum mean pressure was used in data analysis.

Data analysis
Data were analyzed using SPSS 22.0 (IBM Corp., Chicago, IL, USA). Kolmogorov-Smirnov test was used to evaluate data distribution; all data were found to be normally distributed. Descriptive data were presented as mean ± standard deviation. Unpaired t-tests were used to compare PImax and PEmax between sexes. One-way ANOVA with Bonferroni's post hoc adjustment was used to compare respiratory pressures between three age groups. Effect sizes were calculated using Cohen's f for subgroup analysis, classifying as small (< 0.25), moderate (0.25-0.40), and large (> 0.40) [17]. For intergroup analysis, Cohen's d was used, classifying as small (< 0.5), moderate (0.5-0.8), and large (> 0.8) [17]. A stepwise multiple linear regression analysis was performed for PImax and PEmax considering correlations with independent variables (age, weight, and sex). This analysis adds the independent variables to the model and from a significant p-value, we can determine the best model that can predict our dependent variable. Variables that best correlated and made the regression model better explain the variation of the dependent variable were added to the model. In addition, we compared PImax and PEmax values found in this study with respiratory pressures and reference equations determined by previous studies, by obtaining the mean difference between values. Significance level of all tests was set at p < 0.05, and 95% confidence interval was considered. T-test was used based on the mean and standard deviation (SD) of previous values found in the Stefanutti and Fitting study. A power of 99% and α = 0.05 revealed the need for a total of 120 children [18].

Results
One hundred twenty seven children were assessed, of whom 6 were excluded due to inability to perform the respiratory pressure maneuvers. Therefore, 121 children comprised the final sample, with 62 girls. Anthropometric characteristics and pulmonary function are described in Table 1. Table 2 presents the variation of maximum respiratory pressures between sexes and age groups. PImax differed significantly between sexes with a moderate effect size; however, no differences and a small effect size were found for PEmax. There were significant differences between age groups for PImax and PEmax. The two oldest group (8-9 and 10-11 years) presented higher respiratory muscle strength in comparison with children aged 6-7 years. Table 3 presents regression models generated for each maximum respiratory pressure. Independent variables height, weight, age, and sex were positively correlated with PImax, but only age and sex were maintained in the equation. PEmax was positively correlated with height, weight, and age; the variable age was maintained in the equation for girls and weight We calculated the mean difference between maximum respiratory pressures obtained in this study with reference values from previous studies according to sex (Fig 2). These results are similar to the results reported by Heinzmann-Filho et al. and Lanza et al., which also developed reference equations with Brazilian children [8,10]. Table 4 shows the main methodological characteristics of previous studies.
Estimated PImax and PEmax mean values obtained in the present study were also compared with previous Brazilian studies, according to age groups. Prediction lines are drawn in Fig 3A and 3B.

Discussion
We determined new reference values for maximal respiratory pressures in healthy Brazilian children between 6 and 11 years. Previously, several studies [8,9,19,20] indicated reference values for maximal respiratory pressures in healthy children. However, they had limitations such as methodological, and individual variations inherent to the study sample. Pessoa et al., (2014) [7] also followed the recommendations from the ERS and SBPT. They reported that maneuvers methodological standardization and use of digital equipment with software influence maximum mean pressure values (mean maximum pressure sustained for one second) in adults. However, we observed a lack of information regarding maneuvers and equipment, including the software used to choose the best measure in reference values for children. Therefore, it is unspecified whether the interpretation was performed only visually, or some mathematical model of software was incorporated. Graphical visual analysis without mathematical interpretation implies the chance of misinterpretation, leading to an incorrect diagnosis.
This study used a digital device with software to establish reference values for maximum respiratory pressures in healthy children, as recommended by the ERS and SBPT. In this   PEmax, and 8% for PImax and 10% for PEmax, respectively. According to previous studies [2], sexual hormonal differences and greater muscle mass in boy [10] explain the differences between sexes, or even differences in respiratory mechanics, since boys can be more elongated. Respiratory pressures increased with age, and moderate effect sizes were found between groups (6-7, 8-9, and 10-11 years). This increase seems to be linear for both respiratory pressures, but more evident for PImax. Besides differences between age of children included in the studies, we observed a linear relation in most analyzed studies [8,10,11], except for da Rosa et al. (2017) [9], suggesting that age is an important independent variable for reference equations.
Comparing with the present study, most previous studies showed a positive mean difference in maximum pressures with higher respiratory pressures in this sample than in previous studies [9,11,19,20]. The findings were more similar to those reported by Heinzmann-Filho et al. (2012) [8] and Lanza et al. (2015) [10]. Some factors still lead to variations between studies results, namely cultural and regional differences, methodological dissimilarities, types of instruments, and participants' motivation. Heinzmann-Filho et al. (2012) [8] allowed children to place their hands on their cheeks to avoid increasing oral pressure during PEmax. This aspect may lead to variations due to difference in pressure applied by each participant. Different from the recommendation for digital manometers [3], Lanza et al. (2015) [10] used an aneroid manometer, which can under or overestimate pressures measurements.
Age and sex were the best predictors for PImax, age was the best predictor for PEmax in boys, and weight for PEmax in girls. Studies indicate age as the variable that best composes regression models [8,9,19,20], followed by weight [8,9,[20][21][22], and height [9,11,21,22]. Some studies even included body mass index [10,11]  Although some studies followed the recommendation of using digital manometers, the software used to select the largest maneuvers, peak, plateau, and mean pressure was not informed [8,9]. Some studies that used aneroid manometer [10,11] also showed this limitation of not describing how maneuver choice was carried out. These distinctions between studies methodologies may explain differences in respiratory pressures. This study has the advantage of using a digital manometer developed in Brazil (NEPEB, Minas Gerais), with its software for choosing reproducible and acceptable maneuvers that show peak, plateau, and mean pressure values. Although we followed a complete methodology for performing maneuvers using a suitable manometer, it has some limitations. We neither include children from other Brazilian regions nor added evaluations that could interfere with results, such as thorax size and diaphragmatic circumference.
In conclusion, this study determined new reference equations for maximum respiratory pressures in healthy children, including variables such as age, sex, and weight, and using a methodology recommended by ERS and SBPT. Thus, we reinforce the importance of using the recommended methodology, adequate equipment to avoid over or underestimating measures, and to sufficiently report how the maneuvers were selected.